Enzymes have been used within the detergent industry as part of washing formulations for more than 30 years. Proteases are from a commercial perspective the most relevant enzyme in such formulations, but other enzymes including lipases, amylases, cellulases, hemicellulases or mixtures of enzymes are also often used. Proteases are also used in other fields, such as production of diary products, processing of hides, feed processing, etc.
To improve the cost and/or the performance of proteases there is an ongoing search for proteases with altered properties, such as increased activity at low temperatures, increased thermostability, increased specific activity at a given pH, altered Ca2+ dependency, increased stability in the presence of other detergent ingredients (e.g. bleach, surfactants etc.), modified specificity in respect of substrates, etc.
The search for proteases with altered properties includes both discovery of naturally occurring proteases, i.e. so called wild-type proteases but also alteration of well-known proteases by e.g. genetic manipulation of the nucleic acid sequence encoding said proteases. Knowledge of the relationship between the three-dimensional structure and the function of a protein has improved the ability to evaluate which areas of a protein to alter to affect a specific property of the protein.
One group of proteases, which has been indicated for use in detergents, food processing, feed processing is the RP-II proteases or C-component proteases belonging to the protease family S1B, glutamic-acid-specific endopeptidases. This family has till now only received relatively minor attention and has not been further grouped into different sub-groups. However, from the amino acid identities of isolated RP-II proteases it is evident that subgroups exist. Bacillus proteases of the RP-II type are serine proteases that in primary structure are similar to chymotrypsin.
The first description of a protease of the RP-II family of Bacillus proteases was in U.S. Pat. No. 4,266,031 (Tang et al., Novo Industri A/S), where it was designated Component C and tentatively (and incorrectly) characterised as not being a serine protease or metallo protease. Component C was considered a contaminant in the production of the Bacillus licheniformis alkaline protease, subtilisin Carlsberg.
In EP 369 817 (Omnigene Bioproducts, Inc.) the B. subtilis member of the RP-II family was identified by its amino acid and DNA sequences. The enzyme was again stated not to be a serine protease, and the family name RP-II designated (Residual Protease II). The enzyme was characterized further as a metallo protease by the inventors of EP 369 817 (Rufo et al., 1990, J. Bacteriol. 2 1019-1023, and Sloma et al., 1990, J. Bacteriol. 172 1024-1029), designating the enzyme as mpr.
In WO 91/13553 (Novozymes A/S) the amino acid sequence of the C component was disclosed, stating that it is a serine protease specific for glutamic and aspartic acid, while EP 482 879 (Shionogi & Co. Ltd.) disclosed the enzyme and a DNA sequence encoding the C component from B. licheniformis ATCC No. 14580, naming the enzyme BLase. In EP 482 879 the protease is described as being specific for glutamic acid (see also Kakudo et al. “Purification, characterization, cloning, and expression of a glutamic acid-specific protease from Bacillus licheniformis ATCC 14580”. J. Biol. Chem. 267:23782 (1992)).
In 1997 Okamoto et al. (Appl. Microbiol. Biotechnol. (1997) 48 27-33) found that the B. subtilis homologue of BLase, named BSase was identical to the above-mentioned enzyme, mpr/RP-II.
In 1999 Rebrikov et al. (Journal of Protein Chemistry, Vol. 18, No. 1, 1999) disclosed a Glu-specific protease from B. intermedius that also belongs to the RP-II family.
In WO 01/16285 a number of further RP-II protease were disclosed with DNA and amino acid sequences. These RP-II proteases were isolated from B. pumilus, B. halmapalus and B. licheniformis. WO 01/16285 also discloses a number of variants of RP-II proteases. These variants were based on various concepts relating to the primary structure of the RP-II proteases (amino acid sequences).
The homology matrix in Table 1 below clearly indicates that the RP-II proteases 1 to 8 are a distinct group of Glu-specific proteases that are clearly different from the other Glu-specific proteases in the Matrix
TABLE 1123456789101112131100999760555547594645454749210099606059506150444546523100605754476047454544494100949268574438404247510091595444424043456100635339424641457100484141403644810050454646549      10     11    12   13  In the matrix the sequences are identified by the patent publication in which first published or sequence database accession numbers.1. Bacillus sp. JA96 glutamic-acid-specific endopeptidase, JA96, WO 01/162852. 1p3e B. Intermedius, glutamic-acid-specific endopeptidase, BIP, EMBL No. Y5136, Rebrikov et al., Journal of Protein Chemistry, Vol. 18, No. 1, 19993. Bacillus sp. BO32 glutamic-acid-specific endopeptidase, BO32, WO 01/162854. Bacillus licheniformis, BLC, WO 01/16285 (cf. U.S. Pat. No. 4,266,031)5. Bacillus sp. CDJ31 glutamic-acid-specific endopeptidase, CDJ31, WO 01/162856. Bacillus sp. AC116 glutamic-acid-specific endopeptidase, AC116, WO 01/162857. mpr_bacsu Bacillus subtilis serine protease, MPR, EP 369 8178. Bacillus sp. AA513 glutamic-acid-specific endopeptidase, AA513, WO 01/162859. eta_staau Staphylococcus aureus exfoliative toxin A (Lee et al. Sequence determination and comparison of the exfoliative toxin A and toxin B genes from Staphylococcus aureus; J. Bacteriol. 169: 3904 (1987))10. etb_staau Staphylococcus aureus exfoliative toxin B (Jackson, M. P.; Iandolo, J. J.; Sequence of the exfoliative toxin B gene of Staphylococcus aureus; J. Bacteriol. 167: 726 (1986))11. q53781 Staphylococcus aureus (strain Mu50/ATCC 700699) (Rieneck et al.; Submitted (June 1996) to the EMBL/GenBank/DDBJ databases)12. q53782 Staphylococcus aureus (strain Mu50/ATCC 700699) (Rieneck et al., “Molecular cloning and expression of a novel Staphylococcus aureus antigen”. Biochim. Biophys. Acta 1350: 128 (1997)13. stsp_staau Staphylococcal serine endoproteinase V8 Glu-C (Gray, “Nucleotide sequence of the serine protease gene of Staphylococcus aureus, strain V8” Nucleic Acids Res. 15: 6757 (1987)
The three-dimensional structure of the protease Toxin A from Staphylococcus aureus. Belonging to the S1B family has been determined by Cavarelli, J., et al. Structure Vol. 5, p. 813 1997.
However, despite the sequence homology between the proteases belonging to the RP-II proteases and Toxin A from Staphylococcus aureus, modelling of the three-dimensional structure of RP-II proteases on the basis of the three-dimensional structure of Toxin A from Staphylococcus aureus may result in an incorrect three-dimensional structure because of structural differences, especially because the distinct difference in sequence homology to the RP-II proteases.
The inventors of the present invention have elucidated the three-dimensional structure of the C-component protease from Bacillus licheniformis and found that there are several differences between this and the three-dimensional structure of Toxin A from Staphylococcus aureus also belonging to the S1B subgroup of proteases. This surprising difference in structure makes it advantageous to use the BLC structure as basis for homology modelling of RP-II proteases, which, in turn, will improve the ability to obtain desired changes in functionality by protein engineering.